Lupus anticoagulants and antiphospholipid antibodies (APAs) are associated with an increased incidence of thrombosis. Although a causal relationship has not been established, the correlation suggests that these antibodies contribute directly to the thrombotic process. The protein C pathway has been proposed by many, including ourselves, as a possible target for involvement of these APAs. Using a newly developed ELISA assay, we have found that a relatively high percentage of APA patients (20/61) and patients with unexplained thrombosis (20/200) have an antithrombomodulin (TM) antibody at least some of which inhibit protein C activation. Furthermore, our recent finding that optimal activated protein C (APC) anticoagulant activity, but not procoagulant activity, requires the presence of phosphatidylethanolamine (PE) in the membranes provides a potential mechanism to explain selective reduction in APC activity by antibodies that bind to membrane surfaces or protein- membrane complexes. Patients with APAs have been identified whose plasma inhibits APC activity to a greater extent than prothrombin activation and do so in a PE dependent fashion. Preliminary screening suggests that high levels of anti APC activity in APA patients is associated with a history of thrombosis. The goals of the present application are to determine the prevalence of anti-APC and anti-TM activity in APA patients with and without histories of thrombosis and to obtain a molecular understanding of how PE facilitates APC function and participates in APA inhibition of APC activity. This will aid in the design of better assays for detecting APAs with anti-APC activity. The molecular basis of PE function will be approached by determining the nature of PE involvement in facilitating APC binding interactions, by mapping the sites in APC responsible for PE dependent membrane interaction and by determining the mechanisms of inhibition (i.e., direct binding to membranes and the influence of membrane composition vs. binding to protein C or protein S-membrane complexes and whether the antibodies selectively displace APC or protein S), and by using chimeric proteins to map the antigenic determinants. The ability of APAs to inhibit platelet mediated factor Va inactivation vs. prothrombin activation will be examined. Monoclonal antibodies from APA patient B cells will be prepared to study their mechanism of action and potential thrombogenicity. These studies should provide information on the mechanisms by which APAs function, may allow initial insights into which specificities contribute directly to thrombogenicity, and may provide improved assays to monitor the effectiveness of therapeutic approaches.